Accepted Manuscript High diagnostic accuracy and the reproducibility of fine needle aspiration cytology for diagnosing salivary gland tumors: cytohistological correlation in 182 cases Katya Pulido Díaz, DDS, MSc Renê Gerhard, MD, PhD Regina Barros Domingues, MD Leandro Liporoni Martins, MD Ana Carolina Prado Ribeiro, DDS, PhD Márcio Ajudarte Lopes, DDS, PhD Paulo Campos Carneiro, MD, PhD Pablo Agustin Vargas, DDS, PhD, FRCPath PII:
S2212-4403(14)00430-1
DOI:
10.1016/j.oooo.2014.04.004
Reference:
OOOO 902
To appear in:
Oral Surgery, Oral Medicine, Oral Pathology and Oral Radiology
Received Date: 6 November 2013 Revised Date:
28 March 2014
Accepted Date: 8 April 2014
Please cite this article as: Díaz KP, Gerhard R, Domingues RB, Martins LL, Prado Ribeiro AC, Lopes MA, Carneiro PC, Vargas PA, High diagnostic accuracy and the reproducibility of fine needle aspiration cytology for diagnosing salivary gland tumors: cytohistological correlation in 182 cases, Oral Surgery, Oral Medicine, Oral Pathology and Oral Radiology (2014), doi: 10.1016/j.oooo.2014.04.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
*Manuscript Click here to view linked References
ACCEPTED MANUSCRIPT
High diagnostic accuracy and the reproducibility of fine needle aspiration
cytology
for
diagnosing
salivary
gland
tumors:
cytohistological correlation in 182 cases Katya Pulido Díaz, DDS, MSc1; Renê Gerhard, MD, PhD2; Regina Barros Domingues,
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MD2; Leandro Liporoni Martins, MD2; Ana Carolina Prado Ribeiro, DDS, PhD1; Márcio Ajudarte Lopes, DDS, PhD1; Paulo Campos Carneiro, MD, PhD2; Pablo Agustin Vargas, DDS, PhD, FRCPath1* 1
Department of Oral Diagnosis, Oral Pathology, Piracicaba Dental School, State
2
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University of Campinas (FOP-UNICAMP), Piracicaba, São Paulo State, Brazil.
Division of Anatomic Pathology, Hospital das Clínicas, Faculty of Medicine,
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University of São Paulo (HC-FMUSP), São Paulo State, Brazil.
Running title: FNAC and cytohistological correlation in salivary gland tumors.
Abstract word count = 149; Complete manuscript word count = 4054; Number of
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references = 44; number of figures/tables = 9/3
This study was supported by National Council for Scientific and Technological
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Development (CNPq - 302011/2010-2 and PEC-PG - process: 5881102).
DISCLOSURE/DUALITY OF INTEREST
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The authors have no duality of interest to declare. *Corresponding author: Prof. Pablo Agustin Vargas, DDS, MSc, PhD, FRCPath, Oral Diagnosis Department, Piracicaba Dental School, University of Campinas, Avenida Limeira 901, Piracicaba-São Paulo State, Caixa Postal 52, CEP: 13414-903, Brazil. E-mail: [email protected]; Phone: + 55 19 2106-5319; Fax: + 55 19 2106-5218
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ACCEPTED MANUSCRIPT ABSTRACT OBJECTIVES: To assess the efficacy and reproducibility of the cytological diagnosis of salivary gland tumors (SGTs) using fine needle aspiration cytology (FNAC), to
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determine its diagnostic accuracy, sensitivity, specificity, and also to evaluate the extent of interobserver agreement.
STUDY DESIGN: We retrospectively evaluated SGTs from the files of the Division of Pathology at the Clinics Hospital of São Paulo and Piracicaba Dental School between
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2000 and 2006.
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RESULTS: We performed the cytohistological correlation in 182 SGTs. The sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic accuracy were 94%, 100%, 100%, 100% and 99%, respectively. The interobserver cytological reproducibility showed significant statistical concordance (P < 0.0001).
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CONCLUSIONS: FNAC is an effective tool for performing a reliable pre-operative diagnosis in SGTs and shows high diagnostic accuracy and consistent interobserver
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reproducibility. Further FNAC studies analyzing large samples of malignant SGTs and reactive salivary lesions are needed to confirm their accuracy.
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Keywords: cytology, fine-needle aspiration, accuracy, reproducibility, salivary gland tumors, interobserver agreement.
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ACCEPTED MANUSCRIPT INTRODUCTION Salivary gland tumors (SGTs) are a group of complex neoplasms accounting for 3% to 10% of all head and neck tumors.1-4 Fine needle aspiration cytology (FNAC) has been recognized as an important tool for diagnosing benign and malignant nodular lesions in
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thyroid, breast, lymph nodes and salivary glands.5-10
Controversy still exists regarding the clinical indication of FNAC in SGTs because its can cause metaplastic or necrotic changes in the salivary tissue.11,12,13 However, most
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current studies strongly recommend the employment of FNAC in SGTs6,14-16 and have reported significant accuracy (79.1% to 97%), specificity (68.2% to 100%), and
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sensitivity (64% to 94.6%).10,16-21 In our opinion, FNAC will usually show acceptable accuracy in SGTs if performed by experienced cytopathologists. It is also important to note that problems can occur predominantly in the diagnosis of benign or malignant cystic salivary tumors. To the best of our knowledge this is the first paper to assess
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cytological interobserver agreement in SGTs.
Therefore, the objectives of this study were to describe the cytological features of epithelial SGTs, to perform cytohistological correlation in order to access the diagnostic
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accuracy, sensitivity and specificity of FNAC in SGTs, and to assess the interobserver concordance and reproducibility of the cytological diagnosis.
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MATERIAL AND METHODS This study was approved by the Ethics Committee for Human Studies, Piracicaba Dental School, University of Campinas (Protocol #153/2009) and the Ethics Committee for Research Project Analysis, Clinics Hospital of Medicine School, State University of São Paulo (Protocol #223/10). Case selection
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ACCEPTED MANUSCRIPT A total of 242 major SGTs diagnosed by FNAC were collected from two pathology centers: University of Campinas, Piracicaba Dental School (11 cases) and the Clinics Hospital of the University of São Paulo (231 cases) from January 2000 to January 2006. Complete clinical information, which were obtained from the medical records, and
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histopathological slides of the surgical specimens in 182 cases were available. The cytological smears were routinely stained using Diff-Quik and Papanicolaou techniques. Selected cases were also stained using periodic acid-Schiff (PAS). Six of the 242 cases
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and eosin (H&E), and occasionally Alcian blue or PAS.
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had cell blocks available for cytological evaluation and were stained with hematoxylin
All the cases with a complete clinical history, adequate cytological smears and histological slides (surgical specimens of epithelial salivary tumors) were included in the study. If any of these aspects were not present, the case was excluded. It was not goal to evaluate reactive or inflammatory salivary lesions. We excluded 155 non-
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neoplasic lesions: 65 cases of inflammatory diseases (sialadenosis, sialadenitis and granulomatous disease), 49 cystic lesions without atypia (mucous retention cyst,
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lymphoepithelial cystic lesion), 21 lymphoid hyperplasias, 2 lymphangiomas, 1 hemangioma and 17 metastatic diseases (sarcomas and adenocarcinomas).
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Cytologic and histopathologic criteria Cytological smears were reviewed by three cytopathologists (RG, RBD, PAV) and carefully classified into the following categories:
Negative for malignancy: pleomorphic adenoma (PA) and Warthin tumor (WT).
Positive for malignancy: adenoid cystic carcinoma (ACC), mucoepidermoid carcinoma (MEC), acinic cell carcinoma, adenocarcinoma, poorly-differentiated carcinoma, malignant neoplasm with squamous differentiation. 4
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Suspicious: suspicious of well differentiated epithelial neoplasm. Although this diagnosis is clearly a neoplasia it is not possible to define it as benign or malignant only on a cytological basis.
Unsatisfactory: insufficient material.
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The final cytological diagnosis was obtained as a consensus among the three cytopathologists (RG, RBD, PAV) and was only applicable in the cytohistological correlation. The interobserver agreement only considered the individual diagnosis of
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each cytopathologist and the consensus criteria were not included in this analysis.
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The histologic criteria were established by two histopathologists (LL, PAV) who defined them as the "gold standard" strictly based on the criteria of the World Health Organization.22
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Statistical analysis
Based on the histological findings, we categorized the lesions as follows: true negative (TN) when malignancy was not cytologically and histologically present; true positive
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(TP) when both cytological and histological diagnosis were malignant; false negative (FN) when the cytology was interpreted as a benign lesion, but histology was malignant; false positive (FP) when the cytology was interpreted as a malignant
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neoplasm and the histology was benign. A positive predictive value (PPV) means that the probability of a positive result indicates the presence of lesion; a negative predictive value (NPV) means that the probabilty of a negative result indicates the absence of lesion; accuracy is the closeness with which the FNAC approximates the true value and was obtained by comparing the final cytological diagnosis and "gold standard" histopathological diagnosis.
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ACCEPTED MANUSCRIPT To assess interobserver reproducibility, the multiple kappa method was employed.23 The chi-square test was used to determine the correlation of the tumors with age, gender and location, assuming P < 0.05 as indicative of significance. The values of demographic data were obtained by statistical analysis performed using the SAS
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software version 9.2 (SAS Institute Inc., E.U.A., Cary, CN, 2008).
RESULTS
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Of the total of 242 patients, 144 (59.50%) were females and 98 (40.49%) were males with male–female ratio of 1:1.47. Ages ranged from 13 to 94 years with a mean
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age of 49 ± 16.95 years. The parotid gland was the most common site of involvement (190 cases, 78.5%), followed by the submandibular gland (52 cases, 21.4%). Regarding the final cytological diagnosis, 190 (78,5%) were negative for malignancy, 22 (9%) positive for malignancy, 13 (5.3%) suspicious, and 17 (7%)
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unsatisfactory. In the sample, PA (Figures 1A, 1B) was the most frequent diagnosis by FNAC with 155 (64.0%) cases, followed by WT (Figures 1C, 1D) with 35 (14.4%) cases (P < .0001). Among the 22 malignant tumors, 4 (1.6%) were poorly-differentiated
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carcinoma, 4 (1.6%) were carcinoma with squamous differentiation, 4 (1.6%) were adenocarcinoma, 3 (1.2%) were mucoepidermoid carcinoma (MEC) (Figure 2A, 2B), 6 (2.4%) were adenoid cystic carcinoma (ACC) (Figures 2C, 2D), and 1 (0.4%) was
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acinic cell carcinoma (Figure 3A–D). Regarding the 13 cases (5.3%) classified as suspicious, cytological smears displayed well-differentiated epithelial neoplasms (Figure 4A, 4B). The cytological findings for each SGT are depicted in Table I (Figures 5A–D, 6A–D, 7A–D, 9A-B). In this study, 182 out of 242 cases were correlated with the histological diagnosis, which corresponded to 163 benign and 19 malignant SGTs. The cytohistological
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ACCEPTED MANUSCRIPT correlation is summarized in Table II. There were one false-negative case, which presented scarce mixoid stroma and were cytologically diagnosed as PA (Figure 8A), but the surgical specimens exhibited features of carcinoma ex-pleomorphic adenoma (carcinoma ex-PA) (Figure 8B). There were no false-positive diagnoses.
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For the analyis of sensitivity, specificity, PPV, NPV, and diagnostic accuracy, we excluded 11 cases that were cytologically classified as unsatisfactory (n=2) and suspicious (n=7). For the remaining 173 cases, sensitivity, specificity, PPV, NPV, and
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diagnostic accuracy were 94%, 100%, 100%, 100%, 99%, respectively. Considering seven cytological suspicious cases to be malignant, we found an improved sensitivity
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(94%) with the same specificity (96%), but lower values for NPV (96%), PPV (73%), and diagnostic accuracy (96%) (Table III). Regarding interobserver variability, a concordance diagnostic rate of 88% was obtained among the three (RG, RBD, PAV) cytopathologists. After the application of the kappa test, a value of cytological
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interobserver variability of 0.77056 was achieved (P < 0.0001).
DISCUSSION
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Several authors have reported numerous advantages of FNAC, such as that it is unexpensive, fast, efficient, safe, and a well-tolerated technique.10,14,15,17,19,24 In the Clinics Hospital of the University of São Paulo, this diagnostic tool has been used since
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1980 on nodular head and neck lesions. Contributing to the diagnosis and the planning of surgical treatment of SGTs, FNAC has routinely been used in the Oral Diagnosis Clinic at Piracicaba Dental School in the last 12 years. FNAC of salivary gland lesions represents a diagnostic challenge because of the many types of benign and malignant tumors and also the histological variability of each specific tumor type. In spite of this, some authors have published studies including large series of SGTs and detailed
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ACCEPTED MANUSCRIPT cytological descriptions of overlaps and pitfalls, with the purpose of improving diagnostic accuracy.25-28 In our study, the majority of SGTs were benign and PA was the most frequent benign neoplasia, which is in agreement with other studies.2-4,29,30 The most frequent malignant
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tumor in the series was not MEC, as traditionally reported,2,3,29,30 but ACC, which could be explained by the lack of paraffin blocks of MEC in the Hospital’s files. The parotid gland was the most affected site, similar to previous publications.2-4,10,29,30
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It is well-recognized that PA is one of the most easily cytological diagnoses in FNAC because of its classical morphological features.7,31 Even so, we have to be careful
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if atypical cytological features are seen, because these can indicate the presence of carcinoma ex-PA.32,33 We reported two cases of carcinoma ex-PA that were misdiagnosed as PA by FNAC because the cytological features showed plasma cell-like morphology with the presence of scarce myxoid or metachromatic tissue. In fact, these
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were due to sampling errors in which a benign portion of the tumor was aspirated. Similarly, in 2007, Viguer et al.34 reported three cases of carcinoma ex-PA misdiagnosed as benign because the malignant area was not aspirated. To minimize
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misinterpretation, a sufficient sample must be aspirated and the clinical findings should be carefully reviewed.
Diagnosis of WT may be difficult if the cytological aspect mimicks low-grade MEC
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or the smears show squamous metaplasia.35 The regenerative epithelial cells seen in an inflammatory lesion may be misdiagnosed as a WT.14,15 We achieved 100% diagnostic accuracy analyzing 29 cases of WT, which is similar to the study of Flezar and Pogacnick36 who achieved 95% accuracy in 47 cases of WT. ACC is a malignant tumor with high cytological diagnostic accuracy, but there are some features that simulate other SGTs, such as PA, basal cell adenoma, basal cell
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ACCEPTED MANUSCRIPT adenocarcinoma, polymorphous low grade adenocarcinoma, and myoepithelial carcinoma.15,26,37-39 In this study, four cases of ACC diagnosed by FNAC were also confirmed in the surgical specimens. The cytological smears showed small round cells with scant cytoplasm and oval, angulated and hyperchromatic nuclei, as previously
hyaline globules, but they are not pathognomonic of ACC.39
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described by other authors.38,40 Another important cytological feature is the presence of
The five cases histologically diagnosed as high-grade MEC were previously
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cytologically reported as malignant SGTs. Three cases were cytologically diagnosed as carcinoma with squamous differentiation because the smears presented atypical or
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immature squamous cells, but no mucous or glandular cells. One case interpreted as adenocarcinoma showed cell clusters with evident atypia and prominent nuclei in an inflammatory and mucoid background, but there were no squamous or intermediate cells to enable a confident diagnosis of MEC. Another case was diagnosed as a poorly-
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differentiated carcinoma, exhibiting conglomerates of anaplastic epithelial cells, but no mucoid material, or squamous or glandular differentiation. One case cytologically reported as MEC was histologically diagnosed as papillary cystadenocarcinoma. This is
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a rare low-grade carcinoma that represents around 2% of malignant SGTs,29 and few studies have been written describing its cytological features.28,41 Histological examination of the present case revealed large cystic spaces and solid islands composed
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of ovoid to collumnar cells with vacuolated and abundant basophilic cytoplasm. Most of the tumor consisted of a papillary growth pattern and the luminal space was focally filled with mucous material. Acinic cell carcinoma usually shows the presence of atypical large-vacuolated cells with irregular nuclei and granular cytoplasm in a pseudo-acinic duct-like arrangement.42 However, neoplastic acinic cells can be misdiagnosed as oncocytic, mucous cells or
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ACCEPTED MANUSCRIPT even normal acinic cells.25,42,43 In this study, the only case of acinic cell carcinoma was correctly diagnosed by cytology and histological examination, and showed a solid classical pattern. In the present study, the suspicious cytological category fits the terminology of epithelial
neoplasms,"
which
include
tumors
with
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"well-differentiated
cytomorphological findings not sufficient to be classified as malignant. These tumors clearly demonstrated bland cell and architectural features in cytology. Excluding the
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suspicious category from the cytohistological correlation, we found no false-positive diagnoses. However, if we consider the suspicious cases as positives, we obtain a higher
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value of sensitivity (94%), but a significant decrease in PPV (73%) as the majority (6 of 7) of these cases proved to be PA on histological examination. This study also showed a high cytological interobserver reproducibility in diagnosing SGTs. Cell block cytology might play an important role in making a precise diagnosis in
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SGTs and could be a valuable method when there is a sufficient sample to allow it because it may reveal particular histological aspects through staining with H&E, special stains (PAS, Grocott, Ziehl-Neelsen), or immunohistochemistry and in situ
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hybridization. Other techniques, such as core needle biopsy, have been employed on major salivary gland masses and have shown greater accuracy than FNAC. However, in our experience, core needle biopsy can cause serious injuries to the patient, reducing its
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clinical applicability.
The current study had limitations because did not assess reactive or inflammatory lesions in the salivary glands. Further studies comparing the FNAC accuracy between reactive salivary lesions and SGTs can be helpful and in our personal experience FNAC also might be a precise tool to distinguish these lesions.
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ACCEPTED MANUSCRIPT In conclusion, the FNAC technique showed a high diagnostic accuracy and effective interobserver reproducibility in the diagnosis of SGTs. FNAC may be indicated as a reliable tool for the pre-surgical diagnosis of SGTs. It is relevant to mention that the experience and training of the cytopathologist in FNAC play important roles in
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achieving credible results. Further FNAC studies analyzing large samples of malignant
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SGTs and including reactive lesions are needed to confirm accuracy.
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with other salivary myoepithelial tumors. Diagn Cytopathol 2004;31:14-8.
40. Yang GC, Waisman J. Distinguishing adenoid cystic carcinoma from cylindromatous adenomas in salivary fine-needle aspirates: the cytologic clues and
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their ultrastructural basis. Diagn Cytopathol 2006;34:284-8.
41. Aloudah NM, Raddaoui E, Aldhahri S, Al-Abbadi MA. Low-grade papillary
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cystadenocarcinoma of the parotid gland: presentation of a case with cytological, histopathological, and immunohistochemical features and pertinent literature review. Diagn Cytopathol 2009;37:128-31.
42. Klijanienko J, Vielh P. Fine needle sampling of salivary glands lesions V: Cytology
1997;17:347-52.
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of 22 cases of acinic cell carcinoma with histologic correlation. Diagn Cytopathol
43. Al-Khafaji BM, Nestok BR, Katz RL. Fine-needle aspiration of 154 parotid masses
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with histologic correlation: ten-year experience at the University of Texas M. D. Anderson Cancer Center. Cancer 1998;84:153-9. 44. Pfeiffer J, Ridder GJ. Diagnostic value of ultrasound-guided core needle biopsy in
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patients with salivary gland masses. Int J Oral Maxillofac Surg 2012;41:437-43.
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ACCEPTED MANUSCRIPT FIGURE LEGENDS Figure 1A. Pleomorphic adenoma. Cytological smear showing epithelial cells immersed between heavy myxoid metachromatic stroma (Diff-Quik stain, original magnification
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X200). Figure 1B. Pleomorphic adenoma. The cell-block section illustrates evident myoepithelial cells and chondromyxoid stroma in a hemorrhagic background (H&E
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stain, original magnification X200).
Figure 1C. Warthin tumor. The cytological smear shows oncocytic cells with papillary
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arrangement on an intense lymphocythic infiltrate background (Diff-Quik stain, original magnification X200).
Figure 1D. Histologic section of a Warthin tumor demonstrating cystic spaces of oncocytic and columnar epithelial cells surrounded by lymphocytic stroma (H&E stain,
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original magnification X100).
Figure 2A. Low-grade mucoepidermoid carcinoma. Numerous columnar mucous atypic
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cells are showed in a cytologic smear of mucoepidermoid carcinoma (Papanicolaou stain, original magnification X200).
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Figure 2B. Histologic section of a low-grade mucoepidermoid carcinoma showing a cyst growth pattern lined by mucous neoplastic and some intermediate cells (H&E stain, original magnification X200). Figure 2C. Adenoid cystic carcinoma. Cytologic smear showing largely hyaline globules admixed by small round to oval cells with hyperchromatic nuclei (Diff-Quik stain, original magnification X200).
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ACCEPTED MANUSCRIPT Figure 2D. Histologic section showing a cribriform pattern of adenoid cystic carcinoma in a parotid gland (H&E stain, original magnification X100). Figure 3A. Cytological smear of acinic cell carcinoma showing atypical nuclei and
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granular cytoplasm (Diff-Quik stain, original magnification X400). Figure 3B. Cluster of malignant epithelial cells displaying papilliferous and pseudoacinar
arrangement in this cytological smear (Diff-Quik stain, original
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magnification X200).
Figure 3C. Cylindrical PAS-positive structures in a cytological smear of acinic cell
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carcinoma (PAS stain, original magnification X200).
Figure 3D. Histological section of acinic cell carcinoma presenting malignant epithelialacinar cells in a solid type form (H&E stain, original magnification X200).
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Figure 4A. Cytological smear of pleomorphic adenoma composed by epithelial bland tumor cells mixed with hyaline globules and scarce myxoid stroma diagnosed as a suspicious case (Papanicolaou stain X200).
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Figure 4B. Cell-block. Pleomorphic adenoma showing hipercellular area of myoepithelial cells and scarce chondromyxoid stroma in histologic section (H&E
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X200).
Figure 5A. High-grade mucoepidermoid carcinoma. Large clusters of malignant cells immersed in a hemorrhagic background showing squamous cell differentiation in a cytological smear of (Papanicolaou stain X200). Figure 5B. Cytological smear of hypercellular pleomorphic adenoma showing atypical plasmacytoid cells (Diff-Quick stain X400).
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ACCEPTED MANUSCRIPT Figure 5C. Carcinoma ex-pleomorphic adenoma displaying hyperchromatic and poorlydifferentiated epithelial cells with evident hyaline globules and inflammatory/necrotic background (Diff-Quick stain, original magnification 200X).
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Figure 5D. Cytological smear composed by malignant intermediate cells in a scarce mucoid background of mucoepidermoid carcinoma (Papanicolaou stain, original magnification X400).
immersed
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(Diff-Quick
stain,
original
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magnification X400).
background
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Figure 6A. Low-grade mucoepidermoid carcinoma. Pseudohistiocytic mucous cells
Figure 6B. Hexagonal appearance of oncocytic cells in a cytological smear of Warthin Tumor without lymphocytic background (Diff-Quick stain, original magnification X400).
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Figure 6C. Cytological smear of pleomorphic adenoma with abundant myxoid stroma and scattered myoepithelial cells (Papanicolaou stain, original magnification X200).
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Figure 6D. Hypercellular pleomorphic adenoma exhibiting abundant myoepithelial cells mixed with fibrillar stroma (Diff-Quick stain original magnification X200).
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Figure 7A. Papillary cystadenocarcinoma. Observe the cytological smear showing papillary arrangement with atypical vacuolated cytoplasm and evident nuclei dispersed in a clear background (Papanicolaou stain, original magnification X400). Figure 7B. Cytological smear depicting multinucleated malignant epithelial cells of a high-grade mucoepidermoid carcinoma distributed in a dirty background (Diff-Quick stain, original magnification X400).
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ACCEPTED MANUSCRIPT Figure 7C. Large clusters of hyperchromatic small cells surrounded by evident hyaline globules in a cytological smear of adenoid cystic carcinoma (Diff-Quick stain, original magnification X200).
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Figure 7D. Pleomorphic adenoma showing myoepithelial cells with scarce stroma surrounded by small hyaline globules in this cytological smear (Diff-Quick stain, original magnification 200X).
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Figure 8A. Carcinoma ex-pleomorphic adenoma. False negative of a cytologic smear showing epithelial cells on scarce myxoid material and scattered inflammatory cells,
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diagnosed as pleomorphic adenoma (Diff-Quik stain, original magnification X200). Figure 8B. Histologic section of a carcinoma ex-pleomorphic adenoma. Duct-like structures in hyalinized stroma and nest of epithelial atypical hyperchromatic small cells forming cystic spaces (H&E stain, original magnification X200).
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Figure 9A. Scarce neutrophils in a hemorrhagic cytological smear representing an inconclusive case (Diff-Quick stain, original magnification X200).
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Figure 9B. Inconclusive case showing a few histiocytic cells (Diff-Quick stain, original magnification X200).
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Table
ACCEPTED MANUSCRIPT Table 1. Summary of the main cytological features according to the final cytological diagnosis. Cytological features Cellular types
Nuclei
Myoepithelial, spindle, plasmacytoid, epithelial Oncocytic, polygonal with granular cytoplasm, squamousmetaplastic Intermediate, squamous, mucous Myoepithelial, basal, small, scant cytoplasm
Bland, naked or atypical
Round cells, large clear-granular and foamy cytoplasm Ovoidal, round, without cytoplasm
Evident, large, atypical
Adenocarcinoma
Scant cytoplasm, vacuolated
Poorly differentiated carcinoma Malignant neoplasm with squamous differentiation Inconclusive
Fusiform, scant cytoplasm
Finely granular chromatin, evident nucleoli Hyperchromatic, evident nucleoli, mitosis Small, pycnotic, naked
Acinic cell carcinoma
Squamous, hyaline cytoplasm
Without or scant
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Well differentiated epithelial neoplasm
Fig. 1A, 1B, 5B, 6C, 6D, 7D Fig. 1C,1D, 6B
Dispersed, overlapping
Fig. 2A, 2B, 5D, 6A
Clusters, overlapping, cribriform, basaloid
Fig. 2C, 7C,
Dispersed, acinic structures, papilliferous
Fig. 3A, 3B, 3C
Clusters, overlapping
Fig. 4A
Fig. 7A
Inflammatory, dirty, necrosis
Glandular-like structures, overlapping Clusters, overlapping
Inflammatory, necrosis
Clusters, overlapping
Fig. 5A
Proteinaceous, hemorrhagic
Not present
Fig. 9A, 9B
Mucous, dirty
Hyperchomatic, atypia, naked
Cylindrous or finger-like structure, hyaline globules or scant stroma Hyaline, stripelike structure, lymphocytic stroma Without stroma, scattered inflammatory cells Dirty or inflammatory
Not present
Figure reference
Papilliferous, conglomerates
Atypical, polarized
Bland
Cohesive sheets, dispersed
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Adenoid cystic carcinoma
Cellular pattern
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Mucoepidermoid carcinoma
Bland
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Warthin Tumor
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Pleomorphic adenoma
Stromal component or background Chondromyxoid, metachromatic, scarce hyaline globules Lymphoid, proteinaceous, crystalloid formation
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Diagnosis
Fig. 5C, 7B
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ACCEPTED MANUSCRIPT Table 2. Cytohistological correlation in 182 SGTs. Final cytologic diagnosis Pleomorphic adenoma
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Warthin Tumor Adenoid cystic carcinoma Mucoepidermoid carcinoma
n Final histologic diagnosis 128 Pleomorphic adenoma Carcinoma ex-pleomorphic adenoma 29 Warthin tumour 4 Adenoid cystic carcinoma 3 Low-grade mucoepidermoid carcinoma Papilliferous cystadenocarcinoma 3 High-grade mucoepidermoid carcinoma Adenocarcinoma not otherwise specified Poor differentiated squamous cell carcinoma 7 Pleomorphic adenoma Acinic cell carcinoma
Adenocarcinoma
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Suspicious (well differentiated epithelial neoplasm) Poorly differentiated carcinoma Malignant neoplasm with squamous differentiation Inconclusive
1 1 3
2 Low-grade mucoepidermoid carcinoma Pleomorphic adenoma 1 Acinic cell carcinoma 182
1 1 1 182
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2 Carcinoma ex-pleomorphic adenoma High-grade mucoepidermoid carcinoma 3 High-grade mucoepidermoid carcinoma
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Acinic cell carcinoma Total
n 127 1 29 4 2 1 1 1 1 6 1
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Final cytological diagnosis
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Negative for malignancy Positive for malignancy Suspicious Unsatisfactory Total
Benign 156 0 6 1 163
Final histological diagnosis Malignant Total 1 157 16 16 1 7 1 2 19 182
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Table 3. Cytohistological diagnosis correlation for the 182 salivary gland tumors.
S2212-4403(14)00430-1
DOI:
10.1016/j.oooo.2014.04.004
Reference:
OOOO 902
To appear in:
Oral Surgery, Oral Medicine, Oral Pathology and Oral Radiology
Received Date: 6 November 2013 Revised Date:
28 March 2014
Accepted Date: 8 April 2014
Please cite this article as: Díaz KP, Gerhard R, Domingues RB, Martins LL, Prado Ribeiro AC, Lopes MA, Carneiro PC, Vargas PA, High diagnostic accuracy and the reproducibility of fine needle aspiration cytology for diagnosing salivary gland tumors: cytohistological correlation in 182 cases, Oral Surgery, Oral Medicine, Oral Pathology and Oral Radiology (2014), doi: 10.1016/j.oooo.2014.04.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
*Manuscript Click here to view linked References
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High diagnostic accuracy and the reproducibility of fine needle aspiration
cytology
for
diagnosing
salivary
gland
tumors:
cytohistological correlation in 182 cases Katya Pulido Díaz, DDS, MSc1; Renê Gerhard, MD, PhD2; Regina Barros Domingues,
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MD2; Leandro Liporoni Martins, MD2; Ana Carolina Prado Ribeiro, DDS, PhD1; Márcio Ajudarte Lopes, DDS, PhD1; Paulo Campos Carneiro, MD, PhD2; Pablo Agustin Vargas, DDS, PhD, FRCPath1* 1
Department of Oral Diagnosis, Oral Pathology, Piracicaba Dental School, State
2
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University of Campinas (FOP-UNICAMP), Piracicaba, São Paulo State, Brazil.
Division of Anatomic Pathology, Hospital das Clínicas, Faculty of Medicine,
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University of São Paulo (HC-FMUSP), São Paulo State, Brazil.
Running title: FNAC and cytohistological correlation in salivary gland tumors.
Abstract word count = 149; Complete manuscript word count = 4054; Number of
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references = 44; number of figures/tables = 9/3
This study was supported by National Council for Scientific and Technological
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Development (CNPq - 302011/2010-2 and PEC-PG - process: 5881102).
DISCLOSURE/DUALITY OF INTEREST
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The authors have no duality of interest to declare. *Corresponding author: Prof. Pablo Agustin Vargas, DDS, MSc, PhD, FRCPath, Oral Diagnosis Department, Piracicaba Dental School, University of Campinas, Avenida Limeira 901, Piracicaba-São Paulo State, Caixa Postal 52, CEP: 13414-903, Brazil. E-mail: [email protected]; Phone: + 55 19 2106-5319; Fax: + 55 19 2106-5218
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ACCEPTED MANUSCRIPT ABSTRACT OBJECTIVES: To assess the efficacy and reproducibility of the cytological diagnosis of salivary gland tumors (SGTs) using fine needle aspiration cytology (FNAC), to
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determine its diagnostic accuracy, sensitivity, specificity, and also to evaluate the extent of interobserver agreement.
STUDY DESIGN: We retrospectively evaluated SGTs from the files of the Division of Pathology at the Clinics Hospital of São Paulo and Piracicaba Dental School between
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2000 and 2006.
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RESULTS: We performed the cytohistological correlation in 182 SGTs. The sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic accuracy were 94%, 100%, 100%, 100% and 99%, respectively. The interobserver cytological reproducibility showed significant statistical concordance (P < 0.0001).
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CONCLUSIONS: FNAC is an effective tool for performing a reliable pre-operative diagnosis in SGTs and shows high diagnostic accuracy and consistent interobserver
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reproducibility. Further FNAC studies analyzing large samples of malignant SGTs and reactive salivary lesions are needed to confirm their accuracy.
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Keywords: cytology, fine-needle aspiration, accuracy, reproducibility, salivary gland tumors, interobserver agreement.
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ACCEPTED MANUSCRIPT INTRODUCTION Salivary gland tumors (SGTs) are a group of complex neoplasms accounting for 3% to 10% of all head and neck tumors.1-4 Fine needle aspiration cytology (FNAC) has been recognized as an important tool for diagnosing benign and malignant nodular lesions in
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thyroid, breast, lymph nodes and salivary glands.5-10
Controversy still exists regarding the clinical indication of FNAC in SGTs because its can cause metaplastic or necrotic changes in the salivary tissue.11,12,13 However, most
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current studies strongly recommend the employment of FNAC in SGTs6,14-16 and have reported significant accuracy (79.1% to 97%), specificity (68.2% to 100%), and
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sensitivity (64% to 94.6%).10,16-21 In our opinion, FNAC will usually show acceptable accuracy in SGTs if performed by experienced cytopathologists. It is also important to note that problems can occur predominantly in the diagnosis of benign or malignant cystic salivary tumors. To the best of our knowledge this is the first paper to assess
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cytological interobserver agreement in SGTs.
Therefore, the objectives of this study were to describe the cytological features of epithelial SGTs, to perform cytohistological correlation in order to access the diagnostic
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accuracy, sensitivity and specificity of FNAC in SGTs, and to assess the interobserver concordance and reproducibility of the cytological diagnosis.
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MATERIAL AND METHODS This study was approved by the Ethics Committee for Human Studies, Piracicaba Dental School, University of Campinas (Protocol #153/2009) and the Ethics Committee for Research Project Analysis, Clinics Hospital of Medicine School, State University of São Paulo (Protocol #223/10). Case selection
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ACCEPTED MANUSCRIPT A total of 242 major SGTs diagnosed by FNAC were collected from two pathology centers: University of Campinas, Piracicaba Dental School (11 cases) and the Clinics Hospital of the University of São Paulo (231 cases) from January 2000 to January 2006. Complete clinical information, which were obtained from the medical records, and
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histopathological slides of the surgical specimens in 182 cases were available. The cytological smears were routinely stained using Diff-Quik and Papanicolaou techniques. Selected cases were also stained using periodic acid-Schiff (PAS). Six of the 242 cases
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and eosin (H&E), and occasionally Alcian blue or PAS.
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had cell blocks available for cytological evaluation and were stained with hematoxylin
All the cases with a complete clinical history, adequate cytological smears and histological slides (surgical specimens of epithelial salivary tumors) were included in the study. If any of these aspects were not present, the case was excluded. It was not goal to evaluate reactive or inflammatory salivary lesions. We excluded 155 non-
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neoplasic lesions: 65 cases of inflammatory diseases (sialadenosis, sialadenitis and granulomatous disease), 49 cystic lesions without atypia (mucous retention cyst,
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lymphoepithelial cystic lesion), 21 lymphoid hyperplasias, 2 lymphangiomas, 1 hemangioma and 17 metastatic diseases (sarcomas and adenocarcinomas).
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Cytologic and histopathologic criteria Cytological smears were reviewed by three cytopathologists (RG, RBD, PAV) and carefully classified into the following categories:
Negative for malignancy: pleomorphic adenoma (PA) and Warthin tumor (WT).
Positive for malignancy: adenoid cystic carcinoma (ACC), mucoepidermoid carcinoma (MEC), acinic cell carcinoma, adenocarcinoma, poorly-differentiated carcinoma, malignant neoplasm with squamous differentiation. 4
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Suspicious: suspicious of well differentiated epithelial neoplasm. Although this diagnosis is clearly a neoplasia it is not possible to define it as benign or malignant only on a cytological basis.
Unsatisfactory: insufficient material.
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The final cytological diagnosis was obtained as a consensus among the three cytopathologists (RG, RBD, PAV) and was only applicable in the cytohistological correlation. The interobserver agreement only considered the individual diagnosis of
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each cytopathologist and the consensus criteria were not included in this analysis.
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The histologic criteria were established by two histopathologists (LL, PAV) who defined them as the "gold standard" strictly based on the criteria of the World Health Organization.22
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Statistical analysis
Based on the histological findings, we categorized the lesions as follows: true negative (TN) when malignancy was not cytologically and histologically present; true positive
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(TP) when both cytological and histological diagnosis were malignant; false negative (FN) when the cytology was interpreted as a benign lesion, but histology was malignant; false positive (FP) when the cytology was interpreted as a malignant
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neoplasm and the histology was benign. A positive predictive value (PPV) means that the probability of a positive result indicates the presence of lesion; a negative predictive value (NPV) means that the probabilty of a negative result indicates the absence of lesion; accuracy is the closeness with which the FNAC approximates the true value and was obtained by comparing the final cytological diagnosis and "gold standard" histopathological diagnosis.
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ACCEPTED MANUSCRIPT To assess interobserver reproducibility, the multiple kappa method was employed.23 The chi-square test was used to determine the correlation of the tumors with age, gender and location, assuming P < 0.05 as indicative of significance. The values of demographic data were obtained by statistical analysis performed using the SAS
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software version 9.2 (SAS Institute Inc., E.U.A., Cary, CN, 2008).
RESULTS
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Of the total of 242 patients, 144 (59.50%) were females and 98 (40.49%) were males with male–female ratio of 1:1.47. Ages ranged from 13 to 94 years with a mean
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age of 49 ± 16.95 years. The parotid gland was the most common site of involvement (190 cases, 78.5%), followed by the submandibular gland (52 cases, 21.4%). Regarding the final cytological diagnosis, 190 (78,5%) were negative for malignancy, 22 (9%) positive for malignancy, 13 (5.3%) suspicious, and 17 (7%)
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unsatisfactory. In the sample, PA (Figures 1A, 1B) was the most frequent diagnosis by FNAC with 155 (64.0%) cases, followed by WT (Figures 1C, 1D) with 35 (14.4%) cases (P < .0001). Among the 22 malignant tumors, 4 (1.6%) were poorly-differentiated
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carcinoma, 4 (1.6%) were carcinoma with squamous differentiation, 4 (1.6%) were adenocarcinoma, 3 (1.2%) were mucoepidermoid carcinoma (MEC) (Figure 2A, 2B), 6 (2.4%) were adenoid cystic carcinoma (ACC) (Figures 2C, 2D), and 1 (0.4%) was
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acinic cell carcinoma (Figure 3A–D). Regarding the 13 cases (5.3%) classified as suspicious, cytological smears displayed well-differentiated epithelial neoplasms (Figure 4A, 4B). The cytological findings for each SGT are depicted in Table I (Figures 5A–D, 6A–D, 7A–D, 9A-B). In this study, 182 out of 242 cases were correlated with the histological diagnosis, which corresponded to 163 benign and 19 malignant SGTs. The cytohistological
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For the analyis of sensitivity, specificity, PPV, NPV, and diagnostic accuracy, we excluded 11 cases that were cytologically classified as unsatisfactory (n=2) and suspicious (n=7). For the remaining 173 cases, sensitivity, specificity, PPV, NPV, and
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diagnostic accuracy were 94%, 100%, 100%, 100%, 99%, respectively. Considering seven cytological suspicious cases to be malignant, we found an improved sensitivity
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(94%) with the same specificity (96%), but lower values for NPV (96%), PPV (73%), and diagnostic accuracy (96%) (Table III). Regarding interobserver variability, a concordance diagnostic rate of 88% was obtained among the three (RG, RBD, PAV) cytopathologists. After the application of the kappa test, a value of cytological
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interobserver variability of 0.77056 was achieved (P < 0.0001).
DISCUSSION
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Several authors have reported numerous advantages of FNAC, such as that it is unexpensive, fast, efficient, safe, and a well-tolerated technique.10,14,15,17,19,24 In the Clinics Hospital of the University of São Paulo, this diagnostic tool has been used since
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1980 on nodular head and neck lesions. Contributing to the diagnosis and the planning of surgical treatment of SGTs, FNAC has routinely been used in the Oral Diagnosis Clinic at Piracicaba Dental School in the last 12 years. FNAC of salivary gland lesions represents a diagnostic challenge because of the many types of benign and malignant tumors and also the histological variability of each specific tumor type. In spite of this, some authors have published studies including large series of SGTs and detailed
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tumor in the series was not MEC, as traditionally reported,2,3,29,30 but ACC, which could be explained by the lack of paraffin blocks of MEC in the Hospital’s files. The parotid gland was the most affected site, similar to previous publications.2-4,10,29,30
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It is well-recognized that PA is one of the most easily cytological diagnoses in FNAC because of its classical morphological features.7,31 Even so, we have to be careful
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if atypical cytological features are seen, because these can indicate the presence of carcinoma ex-PA.32,33 We reported two cases of carcinoma ex-PA that were misdiagnosed as PA by FNAC because the cytological features showed plasma cell-like morphology with the presence of scarce myxoid or metachromatic tissue. In fact, these
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were due to sampling errors in which a benign portion of the tumor was aspirated. Similarly, in 2007, Viguer et al.34 reported three cases of carcinoma ex-PA misdiagnosed as benign because the malignant area was not aspirated. To minimize
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misinterpretation, a sufficient sample must be aspirated and the clinical findings should be carefully reviewed.
Diagnosis of WT may be difficult if the cytological aspect mimicks low-grade MEC
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or the smears show squamous metaplasia.35 The regenerative epithelial cells seen in an inflammatory lesion may be misdiagnosed as a WT.14,15 We achieved 100% diagnostic accuracy analyzing 29 cases of WT, which is similar to the study of Flezar and Pogacnick36 who achieved 95% accuracy in 47 cases of WT. ACC is a malignant tumor with high cytological diagnostic accuracy, but there are some features that simulate other SGTs, such as PA, basal cell adenoma, basal cell
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hyaline globules, but they are not pathognomonic of ACC.39
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described by other authors.38,40 Another important cytological feature is the presence of
The five cases histologically diagnosed as high-grade MEC were previously
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cytologically reported as malignant SGTs. Three cases were cytologically diagnosed as carcinoma with squamous differentiation because the smears presented atypical or
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immature squamous cells, but no mucous or glandular cells. One case interpreted as adenocarcinoma showed cell clusters with evident atypia and prominent nuclei in an inflammatory and mucoid background, but there were no squamous or intermediate cells to enable a confident diagnosis of MEC. Another case was diagnosed as a poorly-
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differentiated carcinoma, exhibiting conglomerates of anaplastic epithelial cells, but no mucoid material, or squamous or glandular differentiation. One case cytologically reported as MEC was histologically diagnosed as papillary cystadenocarcinoma. This is
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a rare low-grade carcinoma that represents around 2% of malignant SGTs,29 and few studies have been written describing its cytological features.28,41 Histological examination of the present case revealed large cystic spaces and solid islands composed
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of ovoid to collumnar cells with vacuolated and abundant basophilic cytoplasm. Most of the tumor consisted of a papillary growth pattern and the luminal space was focally filled with mucous material. Acinic cell carcinoma usually shows the presence of atypical large-vacuolated cells with irregular nuclei and granular cytoplasm in a pseudo-acinic duct-like arrangement.42 However, neoplastic acinic cells can be misdiagnosed as oncocytic, mucous cells or
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ACCEPTED MANUSCRIPT even normal acinic cells.25,42,43 In this study, the only case of acinic cell carcinoma was correctly diagnosed by cytology and histological examination, and showed a solid classical pattern. In the present study, the suspicious cytological category fits the terminology of epithelial
neoplasms,"
which
include
tumors
with
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"well-differentiated
cytomorphological findings not sufficient to be classified as malignant. These tumors clearly demonstrated bland cell and architectural features in cytology. Excluding the
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suspicious category from the cytohistological correlation, we found no false-positive diagnoses. However, if we consider the suspicious cases as positives, we obtain a higher
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value of sensitivity (94%), but a significant decrease in PPV (73%) as the majority (6 of 7) of these cases proved to be PA on histological examination. This study also showed a high cytological interobserver reproducibility in diagnosing SGTs. Cell block cytology might play an important role in making a precise diagnosis in
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SGTs and could be a valuable method when there is a sufficient sample to allow it because it may reveal particular histological aspects through staining with H&E, special stains (PAS, Grocott, Ziehl-Neelsen), or immunohistochemistry and in situ
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hybridization. Other techniques, such as core needle biopsy, have been employed on major salivary gland masses and have shown greater accuracy than FNAC. However, in our experience, core needle biopsy can cause serious injuries to the patient, reducing its
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clinical applicability.
The current study had limitations because did not assess reactive or inflammatory lesions in the salivary glands. Further studies comparing the FNAC accuracy between reactive salivary lesions and SGTs can be helpful and in our personal experience FNAC also might be a precise tool to distinguish these lesions.
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ACCEPTED MANUSCRIPT In conclusion, the FNAC technique showed a high diagnostic accuracy and effective interobserver reproducibility in the diagnosis of SGTs. FNAC may be indicated as a reliable tool for the pre-surgical diagnosis of SGTs. It is relevant to mention that the experience and training of the cytopathologist in FNAC play important roles in
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achieving credible results. Further FNAC studies analyzing large samples of malignant
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SGTs and including reactive lesions are needed to confirm accuracy.
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37. Lee SS, Cho KJ, Jang JJ, Ham EK. Differential diagnosis of adenoid cystic carcinoma from pleomorphic adenoma of the salivary gland on fine needle aspiration cytology. Acta Cytol 1996;40:1246-52. 38. Klijanienko J, Vielh P. Fine-needle sampling of salivary gland lesions. III. Cytologic and histologic correlation of 75 cases of adenoid cystic carcinoma:
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ACCEPTED MANUSCRIPT review and experience at the Institut Curie with emphasis on cytologic pitfalls. Diagn Cytopathol 1997;17:36-41. 39. Kawahara A, Harada H, Kage M, Yokoyama T, Kojiro M. Extracellular material in adenoid cystic carcinoma of the salivary glands: a comparative cytological study
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with histologic correlation: ten-year experience at the University of Texas M. D. Anderson Cancer Center. Cancer 1998;84:153-9. 44. Pfeiffer J, Ridder GJ. Diagnostic value of ultrasound-guided core needle biopsy in
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patients with salivary gland masses. Int J Oral Maxillofac Surg 2012;41:437-43.
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ACCEPTED MANUSCRIPT FIGURE LEGENDS Figure 1A. Pleomorphic adenoma. Cytological smear showing epithelial cells immersed between heavy myxoid metachromatic stroma (Diff-Quik stain, original magnification
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X200). Figure 1B. Pleomorphic adenoma. The cell-block section illustrates evident myoepithelial cells and chondromyxoid stroma in a hemorrhagic background (H&E
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stain, original magnification X200).
Figure 1C. Warthin tumor. The cytological smear shows oncocytic cells with papillary
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arrangement on an intense lymphocythic infiltrate background (Diff-Quik stain, original magnification X200).
Figure 1D. Histologic section of a Warthin tumor demonstrating cystic spaces of oncocytic and columnar epithelial cells surrounded by lymphocytic stroma (H&E stain,
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original magnification X100).
Figure 2A. Low-grade mucoepidermoid carcinoma. Numerous columnar mucous atypic
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cells are showed in a cytologic smear of mucoepidermoid carcinoma (Papanicolaou stain, original magnification X200).
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Figure 2B. Histologic section of a low-grade mucoepidermoid carcinoma showing a cyst growth pattern lined by mucous neoplastic and some intermediate cells (H&E stain, original magnification X200). Figure 2C. Adenoid cystic carcinoma. Cytologic smear showing largely hyaline globules admixed by small round to oval cells with hyperchromatic nuclei (Diff-Quik stain, original magnification X200).
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ACCEPTED MANUSCRIPT Figure 2D. Histologic section showing a cribriform pattern of adenoid cystic carcinoma in a parotid gland (H&E stain, original magnification X100). Figure 3A. Cytological smear of acinic cell carcinoma showing atypical nuclei and
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granular cytoplasm (Diff-Quik stain, original magnification X400). Figure 3B. Cluster of malignant epithelial cells displaying papilliferous and pseudoacinar
arrangement in this cytological smear (Diff-Quik stain, original
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Figure 3C. Cylindrical PAS-positive structures in a cytological smear of acinic cell
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carcinoma (PAS stain, original magnification X200).
Figure 3D. Histological section of acinic cell carcinoma presenting malignant epithelialacinar cells in a solid type form (H&E stain, original magnification X200).
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Figure 4A. Cytological smear of pleomorphic adenoma composed by epithelial bland tumor cells mixed with hyaline globules and scarce myxoid stroma diagnosed as a suspicious case (Papanicolaou stain X200).
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Figure 4B. Cell-block. Pleomorphic adenoma showing hipercellular area of myoepithelial cells and scarce chondromyxoid stroma in histologic section (H&E
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X200).
Figure 5A. High-grade mucoepidermoid carcinoma. Large clusters of malignant cells immersed in a hemorrhagic background showing squamous cell differentiation in a cytological smear of (Papanicolaou stain X200). Figure 5B. Cytological smear of hypercellular pleomorphic adenoma showing atypical plasmacytoid cells (Diff-Quick stain X400).
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ACCEPTED MANUSCRIPT Figure 5C. Carcinoma ex-pleomorphic adenoma displaying hyperchromatic and poorlydifferentiated epithelial cells with evident hyaline globules and inflammatory/necrotic background (Diff-Quick stain, original magnification 200X).
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Figure 5D. Cytological smear composed by malignant intermediate cells in a scarce mucoid background of mucoepidermoid carcinoma (Papanicolaou stain, original magnification X400).
immersed
in
a
proteinaceous-cystic
(Diff-Quick
stain,
original
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background
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Figure 6A. Low-grade mucoepidermoid carcinoma. Pseudohistiocytic mucous cells
Figure 6B. Hexagonal appearance of oncocytic cells in a cytological smear of Warthin Tumor without lymphocytic background (Diff-Quick stain, original magnification X400).
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Figure 6C. Cytological smear of pleomorphic adenoma with abundant myxoid stroma and scattered myoepithelial cells (Papanicolaou stain, original magnification X200).
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Figure 6D. Hypercellular pleomorphic adenoma exhibiting abundant myoepithelial cells mixed with fibrillar stroma (Diff-Quick stain original magnification X200).
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Figure 7A. Papillary cystadenocarcinoma. Observe the cytological smear showing papillary arrangement with atypical vacuolated cytoplasm and evident nuclei dispersed in a clear background (Papanicolaou stain, original magnification X400). Figure 7B. Cytological smear depicting multinucleated malignant epithelial cells of a high-grade mucoepidermoid carcinoma distributed in a dirty background (Diff-Quick stain, original magnification X400).
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ACCEPTED MANUSCRIPT Figure 7C. Large clusters of hyperchromatic small cells surrounded by evident hyaline globules in a cytological smear of adenoid cystic carcinoma (Diff-Quick stain, original magnification X200).
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Figure 7D. Pleomorphic adenoma showing myoepithelial cells with scarce stroma surrounded by small hyaline globules in this cytological smear (Diff-Quick stain, original magnification 200X).
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Figure 8A. Carcinoma ex-pleomorphic adenoma. False negative of a cytologic smear showing epithelial cells on scarce myxoid material and scattered inflammatory cells,
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diagnosed as pleomorphic adenoma (Diff-Quik stain, original magnification X200). Figure 8B. Histologic section of a carcinoma ex-pleomorphic adenoma. Duct-like structures in hyalinized stroma and nest of epithelial atypical hyperchromatic small cells forming cystic spaces (H&E stain, original magnification X200).
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Figure 9A. Scarce neutrophils in a hemorrhagic cytological smear representing an inconclusive case (Diff-Quick stain, original magnification X200).
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Figure 9B. Inconclusive case showing a few histiocytic cells (Diff-Quick stain, original magnification X200).
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Fig. 1A; Fig. 1B; Fig. 1C; Fig. 1D. Click here to download high resolution image
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Table
ACCEPTED MANUSCRIPT Table 1. Summary of the main cytological features according to the final cytological diagnosis. Cytological features Cellular types
Nuclei
Myoepithelial, spindle, plasmacytoid, epithelial Oncocytic, polygonal with granular cytoplasm, squamousmetaplastic Intermediate, squamous, mucous Myoepithelial, basal, small, scant cytoplasm
Bland, naked or atypical
Round cells, large clear-granular and foamy cytoplasm Ovoidal, round, without cytoplasm
Evident, large, atypical
Adenocarcinoma
Scant cytoplasm, vacuolated
Poorly differentiated carcinoma Malignant neoplasm with squamous differentiation Inconclusive
Fusiform, scant cytoplasm
Finely granular chromatin, evident nucleoli Hyperchromatic, evident nucleoli, mitosis Small, pycnotic, naked
Acinic cell carcinoma
Squamous, hyaline cytoplasm
Without or scant
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Well differentiated epithelial neoplasm
Fig. 1A, 1B, 5B, 6C, 6D, 7D Fig. 1C,1D, 6B
Dispersed, overlapping
Fig. 2A, 2B, 5D, 6A
Clusters, overlapping, cribriform, basaloid
Fig. 2C, 7C,
Dispersed, acinic structures, papilliferous
Fig. 3A, 3B, 3C
Clusters, overlapping
Fig. 4A
Fig. 7A
Inflammatory, dirty, necrosis
Glandular-like structures, overlapping Clusters, overlapping
Inflammatory, necrosis
Clusters, overlapping
Fig. 5A
Proteinaceous, hemorrhagic
Not present
Fig. 9A, 9B
Mucous, dirty
Hyperchomatic, atypia, naked
Cylindrous or finger-like structure, hyaline globules or scant stroma Hyaline, stripelike structure, lymphocytic stroma Without stroma, scattered inflammatory cells Dirty or inflammatory
Not present
Figure reference
Papilliferous, conglomerates
Atypical, polarized
Bland
Cohesive sheets, dispersed
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Adenoid cystic carcinoma
Cellular pattern
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Mucoepidermoid carcinoma
Bland
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Warthin Tumor
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Pleomorphic adenoma
Stromal component or background Chondromyxoid, metachromatic, scarce hyaline globules Lymphoid, proteinaceous, crystalloid formation
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Diagnosis
Fig. 5C, 7B
Table
ACCEPTED MANUSCRIPT Table 2. Cytohistological correlation in 182 SGTs. Final cytologic diagnosis Pleomorphic adenoma
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Warthin Tumor Adenoid cystic carcinoma Mucoepidermoid carcinoma
n Final histologic diagnosis 128 Pleomorphic adenoma Carcinoma ex-pleomorphic adenoma 29 Warthin tumour 4 Adenoid cystic carcinoma 3 Low-grade mucoepidermoid carcinoma Papilliferous cystadenocarcinoma 3 High-grade mucoepidermoid carcinoma Adenocarcinoma not otherwise specified Poor differentiated squamous cell carcinoma 7 Pleomorphic adenoma Acinic cell carcinoma
Adenocarcinoma
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Suspicious (well differentiated epithelial neoplasm) Poorly differentiated carcinoma Malignant neoplasm with squamous differentiation Inconclusive
1 1 3
2 Low-grade mucoepidermoid carcinoma Pleomorphic adenoma 1 Acinic cell carcinoma 182
1 1 1 182
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2 Carcinoma ex-pleomorphic adenoma High-grade mucoepidermoid carcinoma 3 High-grade mucoepidermoid carcinoma
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Acinic cell carcinoma Total
n 127 1 29 4 2 1 1 1 1 6 1
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Final cytological diagnosis
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Negative for malignancy Positive for malignancy Suspicious Unsatisfactory Total
Benign 156 0 6 1 163
Final histological diagnosis Malignant Total 1 157 16 16 1 7 1 2 19 182
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Table 3. Cytohistological diagnosis correlation for the 182 salivary gland tumors.
